The present invention relates generally to the use of electric pulses to increase the permeability of a cell and more specifically to the introduction of nucleic acids into a cell using electric pulses at a low electric field strength for a long pulse length.
The ability to introduce foreign DNA into host cells on one of the principal tools of molecular biology. There are five general types of methods for transfecting eukaryotic host cells: (1) direct introduction of cloned DNA by microinjection, (2) use of viral vectors, (3) encapsulation in a carrier system such as a liposome, (4) the use of facilitators such as calcium phosphate or diethylaminoetyl (DEAE) dextran, and (5) electroporation. Although all of these methods allow the transfer of DNA into dividing cells, fewer methods are available for the transfer of DNA into non-dividing cells.
In the 1970""s it was discovered that electric fields could be used to create pores in cells without causing permanent damage. This discovery made possible the insertion of large molecules into cell cytoplasm. It is known that genes and other molecules such as pharmacological compounds can be incorporated into live cells through a process known as electroporation. The genes or other molecules are mixed with the live cells in a buffer medium and short pulses of high electric fields are applied. The cell membranes are transiently made porous and the genes or molecules enter the cells, where they can modify the genome of the cell.
Studies have shown that large size nucleotide sequences (up to 630 kb) can be introduced into mammalian cells via electroporation (Eanault, et al., Gene (Amsterdam), 144(2):205, 1994; Nucleic Acids Research, 15(3): 1311, 1987; Knutson, et al., Anal. Biochem., 164:44, 1987; Gibson, et al., EMBO J., 6(8):2457, 1987; Dower, et al., Genetic Engineering, 12:275, 1990; Mozo, et al., Plant Molecular Biology, 16:917, 1991). However, the efficiency of electroporation, as reflected in the current literature, is low (see U.S. Pat. No. 5,019,034, herein incorporated by reference). A typical result is from 5 to 20 percent transfection depending on conditions, parameters and cell type. Creation of a high efficiency method for the transfer of nucleic acid via electroporation would make the method useful for the introduction of nucleic acid into cells both in vivo and in vitro.
The present invention provides a method for introducing nucleic acid into a cell by contacting the cell with a nucleic acid and applying a low voltage electrical impulse for a long pulse length. The electrical impulse is of sufficient duration and strength to introduce the nucleic acid into the cell. The method can be utilized in vitro or in vivo. The cells can be dividing or non-dividing.
The invention also provides a method for introducing polypeptides into a cell by contacting the cell with a polypeptide and applying a low electrical field impulse for a long pulse length. The electrical impulse is of sufficient duration and strength to introduce the polypeptide into the cell. The method can be utilized in vitro or in vivo. The cells can be dividing or non-dividing.